Role of dysregulated miRNA in Tregs in Multiple Sclerosis

Tregs 中失调的 miRNA 在多发性硬化症中的作用

• 批准号: 8463637
• 负责人: Amy E Lovett-Racke
• 金额: $ 18.4万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463637
• 关键词: 3' Untranslated Regions; Affect; Algorithms; Autoimmune Diseases; Biological Assay; CD4 Positive T Lymphocytes; Cell Differentiation process; Cells; Characteristics; Defect; Development; Disease; Disease Progression; Disease susceptibility; Environment; Equilibrium; Figs - dietary; Functional disorder; Gene Expression; Gene Targeting; Genes; Health; Human; Immune; Immune system; Inflammatory; Laboratories; Luciferases; Maintenance; Mediating; MicroRNAs; Multiple Sclerosis; Nerve Degeneration; Neurologic; Pathology; Pathway interactions; Patients; Phenotype; Play; Predisposition; Production; Proteins; RNA Interference Pathway; Regulatory T-Lymphocyte; Relative (related person); Role; Signal Pathway; Signal Transduction; Sorting - Cell Movement; T-Lymphocyte; Th1 Cells; Th2 Cells; Work; central nervous system demyelinating disorder; cytokine; disability; in vivo; inhibitor/antagonist; innovation; memory CD4 T lymphocyte; novel; peripheral blood; receptor; young adult

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. My laboratory has performed a large miRNA profiling study on naive and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. The TGF¿ signaling pathway was predicted to be suppressed in MS patients by 12 miRNAs identified as being dysregulated in CD4 T cells. TGF¿ has shown to play a vital role in CD4+CD25hiFoxp3+ regulatory T cells (Tregs), and Tregs have been shown to be defective in MS patients. TGF¿ signaling has been shown to be critical for production of iTregs, and the maintenance of suppressor functions and Foxp3 expression in Tregs. We hypothesize that dysregulated miRNA expression in CD4 T cells in MS patients' results in impaired TGF¿ signaling and defective CD4 regulatory T cells, promoting the onset and progression of MS. Aim 1: To determine if miRNAs identified as dysregulated in CD4 T cells in MS patients directly target specific molecules in the TGF ¿ signaling pathway. First, we will determine if there is a correlation in gene expression levels between specific miRNAs and predicted TGF¿ targets in human CD4 T cells from MS patients and healthy controls. Second, we will use luciferase assays to determine if specific miRNAs suppress the 3' UTR sequence of the predicted target genes in the TGF¿ pathway. Aim 2: To determine if the miRNAs that target the TGF¿ pathway alter iTreg development and/or suppressive functions of Tregs. TGF¿ targeting miRNAs will be inhibited or over-expressed in naive CD4 T cells and then cultured under iTreg conditions. The resulting T cells will be phenotyped for iTreg characteristics. In addition, Tregs will be transfected with specific miRNA inhibitors, evaluated for sensitivity to TGF¿, and ability to suppress effector T cells. Aim 3: To determine if there is an association between the level of TGF¿-associated miRNA expression and the number of Tregs in vivo, or an association between TGF¿-associated miRNA expression and suppressive capacity of Tregs in MS patients. Flow cytometric analysis of peripheral blood CD4 T cells from MS patients will evaluate the number of Tregs in MS patients relative to the expression of TGF¿-associated miRNAs. In addition, Tregs will be sorted from peripheral blood of MS patients, evaluated for suppressive capacity, and determine if there is a correlation between TGF¿-associated miRNAs and Treg number and/or function. Given the well-documented role of Tregs in maintaining tolerance to self and the observation that CD4 Tregs are defective in MS patients, this novel study will determine if dysregulated miRNA expression in CD4 T cells underlies this Treg defect in MS patients. Although several studies have identified miRNAs that play a role in Tregs17-22, no studies have identified differentially expressed miRNAs in MS patients that are specifically associated with genes in the TG¿ pathway and their influence on Tregs in an autoimmune disease. Treg cells play an important role in the delicate balance of protection from our environment and protecting ourselves from our own immune system, and TGF¿ has pleiotrophic effects that extend beyond Tregs that have vast importance in health and disease, making this an invaluable study.

描述(申请人提供)：多发性硬化症(MS)是一种免疫介导的中枢神经系统(CNS)脱髓鞘疾病，可导致严重的神经功能障碍。疾病的原因尚不清楚，疾病病理生理学基础上的免疫和神经退行性机制知之甚少，目前的治疗方法在减缓疾病进展方面只有部分有效。我的实验室对未经治疗的MS患者的幼稚和效应/记忆的CD4T细胞进行了大规模的miRNA图谱研究，以确定miRNA是否可能有助于疾病的易感性或进展。据预测，在MS患者中，12个在CD4T细胞中调控异常的miRNAs抑制了转化生长因子β信号通路。已证明转化生长因子在CD4+CD25hiFoxp3+调节性T细胞(Tregs)中起着至关重要的作用，而Tregs在MS患者中被证明是有缺陷的。已经证明，转化生长因子信号转导对于iTregs的产生、抑制功能的维持和Tregs中Foxp3的表达是至关重要的。我们假设MS患者CD4T细胞中miRNA的表达异常导致了转化生长因子β信号转导受损和CD4调节T细胞缺陷，从而促进了多发性硬化的发生和发展。目的1：确定MS患者CD4T细胞中表达异常的miRNAs是否直接靶向转化生长因子β信号通路中的特定分子。首先，我们将确定MS患者和健康对照组的人类CD4T细胞中特定miRNAs和预测的转化生长因子β靶标之间的基因表达水平是否存在相关性。其次，我们将使用荧光素酶分析来确定特定的miRNAs是否抑制了转化生长因子途径中预测的靶基因的3‘UTR序列。目的2：确定靶向转化生长因子途径的miRNAs是否改变了tregs的iTreg发育和/或抑制功能。靶向miRNAs的转化生长因子在初始的CD4T细胞中被抑制或过度表达，然后在iTreg条件下培养。生成的T细胞将根据iTreg特征进行表型分析。此外，Tregs将被导入特定的miRNA抑制剂，评估其对转化生长因子的敏感性和抑制效应性T细胞的能力。目的：探讨多发性硬化症患者体内转化生长因子相关miRNA表达水平与Tregs数量之间的关系，以及转化生长因子相关miRNA表达与Tregs抑制能力之间的关系。对MS患者外周血中CD4T细胞的流式细胞仪分析将评估MS患者中Tregs的数量与转化生长因子相关miRNAs的表达。此外，将从MS患者的外周血中分离Treg，评估其抑制能力，并确定与转化生长因子相关的miRNAs与Treg数量和/或功能之间是否存在相关性。鉴于Tregs在维持对自身的耐受性方面的作用已经得到了充分的证明，并且观察到MS患者的CD4Tregs存在缺陷，这项新的研究将确定CD4T细胞中的miRNA表达异常是否是MS患者Treg缺陷的基础。尽管一些研究已经确定了在Tregs17-22中发挥作用的miRNAs，但还没有研究发现在MS患者中差异表达的miRNAs与TG途径中的基因及其在自身免疫性疾病中对Tregs的影响有关。Treg细胞在保护我们的环境和保护我们自己的免疫系统之间的微妙平衡中扮演着重要的角色，而且转化生长因子？具有超出Treg的多重营养作用，对健康和疾病具有极其重要的作用，使这项研究成为一项无价的研究。